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In response to the market demand for therapeutic antibodies, the upstream cell culture scale and expression titer of antibodies have been significantly improved, while the production efficiency of downstream purification process is relatively fall behind, and the downstream processing capacity has become a bottleneck limiting antibody production throughput. Using monoclonal antibody mab-X as experimental material, we optimized the caprylic acid (CA) precipitation process conditions of cell culture fluid and low pH virus inactivation pool, and studied two applications of using CA treatment to remove aggregates and to inactivate virus. Based on the lab scale study, we carried out a 500 L scale-up study, where CA was added to the low pH virus inactivation pool for precipitation, and the product quality and yield before and after precipitation were detected and compared. We found that CA precipitation significantly reduced HCP residuals and aggregates both before and after protein A affinity chromatography. In the aggregate spike study, CA precipitation removed about 15% of the aggregates. A virus reduction study showed complete clearance of a model retrovirus during CA precipitation of protein A purified antibody. In the scale-up study, the depth filtration harvesting, affinity chromatography, low pH virus inactivation, CA precipitation and depth filtration, and cation exchange chromatography successively carried out. The mixing time and stirring speed in the CA precipitation process significantly affected the CA precipitation effect. After CA precipitation, the HCP residue in the low pH virus inactivation solution decreased 895 times. After precipitation, the product purity and HCP residual meet the quality criteria of monoclonal antibodies. CA precipitation can reduce the chromatography step in the conventional purification process. In conclusion, CA precipitation in the downstream process can simplify the conventional purification process, fully meet the purification quality criterion of mab-X, and improve production efficiency and reduce production costs. The results of this study may promote the application of CA precipitation in the purification of monoclonal antibodies, and provide a reference for solving the bottleneck of the current purification process.
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Cricetinae , Animals , Antibodies, Monoclonal/metabolism , Caprylates/chemistry , Cell Culture Techniques , Chromatography, Affinity , CHO Cells , Cricetulus , Chemical PrecipitationABSTRACT
Abstract The aim of this scoping review was to provide sufficient information about the effectiveness of ozone gas in virus inactivation of surfaces and objects under different environmental conditions. The review was performed according to the list of PRISMA SrC recommendations and the JBI Manual for Evidence Synthesis for Scoping Reviews. The review was registered in Open Science Framework (OSF). EMBASE (Ovid), Lilacs, LIVIVO, MEDLINE (PubMed), SciELO, Scopus and Web of Science were primary sources, and "gray literature" was searched in OpenGray and OpenThesis. A study was included if it reported primary data on the effect of ozone gas application for vehicle-borne and airborne virus inactivation. No language or publication date restriction was applied. The search was conduct on July 1, 2020. A total of 16,120 studies were screened, and after exclusion of noneligible studies, fifteen studies fulfilled all selection criteria. Application of ozone gas varied in terms of concentration, ozone exposure period and the devices used to generate ozone gas. Twelve studies showed positive results for inactivation of different virus types, including bacteriophages, SARS-CoV-2 surrogates and other vehicle-borne viruses. Most of the studies were classified as unclear regarding sponsorship status. Although most of the population has not yet been vaccinated against COVID-19, disinfection of environments, surfaces, and objects is an essential prevention strategy to control the spread of this disease. The results of this Scoping Review demonstrate that ozone gas is promising for viral disinfection of surfaces.
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【Objective】 To establish a simple, economical and rapid method for the determination of methylene blue (MB) release in virus inactivation bag. 【Methods】 Based on the fluorescence energy transfer between MB and BSA-stabilized gold nanoclusters (BSA-AuNCs), the standard curve of MB determination was established by measuring the fluorescence quenching degree of MB to BSA-AuNCs in different concentrations to conduct the determination of MB release in virus inactivation bag. 【Results】 There was a good linear relationship between the MB concentration (cMB) and the fluorescence quenching degree of BSA-AuNCs[ (I0-I)/I0=0.018cMB+ 0.021(r=0.996)] when the fluorescence emission wavelength was about 620 nm and the cMB was in the range of (0.9-36) μmoL/L. The recovery of MB was 98.00% -101.95 % when applied to determine MB at high, medium, and low concentrations, the obtained intra-day variation coefficients were 0.73%, 0.81% and 0.77% respectively, and the obtained inter-day variation coefficients were 3.92%, 3.81%, and 4.73% respectively. There was no significant difference between the results measured by this method and those measured by combination of solid-phase extraction and spectrophotometry(P>0.05). 【Conclusion】 The fluorescence energy transfer method could achieve simple and rapid determination of MB release in virus inactivation bag with accurate and reliable results.
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【Objective】 To investigate the effect of sample processing at 56℃ for 30 min on routine examination in Department of Blood Transfusion. 【Methods】 A total of 40 cross matched blood samples submitted by clinical departments of our hospital, were collected, and each sample was equally divided into two. Before and after heating at 56℃ for 30 min, the ABO blood group was detected by manual method and card method (gel card and glass beadle card), antibody titer was detected by coagulant method, and cross-matching was conducted by anti-globulin card method. Chi-square test and Wilcoxon signed rank test were used to compare the differences between the two groups (before and after heating treatment). 【Results】 The blood group detection rates of the experimental group were 100% (40/40), 37.5% (15/40) and 80% (32/40) by manual test tube method, gel card and glass beads card, respectively, P0.05). The matching rate of two groups of samples, cross-matched with corresponding donor samples, was both 100% (40/40) by coagulant method, and 100% (40/40) vs 25% (10/40) respectively by the antiglobulin card method (P<0.01). The other 30 samples in the experimental group presented weak agglutination in the secondary side. 【Conclusion】 The treatment of virus inactivation at 56℃ for 30 min has little effect on blood group identification by test tube method, antibody titer and cross-matching by coagulant method, and reduceds the occupational exposure of staff in Blood Transfusion Department.
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OBJECTIVE：To study the effects of virus in activation treatment of plasma specimen on plasma concentration determination of voriconzole ，linezolid，vancomycin and teicoplanin. METHODS ：The remaining plasma of 36 inpatients in our hospital after routine blood concentration examination of voriconazole ，linezolid，vancomycin and teicoplanin were collected as specimen（9 drug-contained plasma specimens for each drug ），and merged into three different concentration levels （low，medium， high）of mixed samples according the results of routine blood test. Then the mixed samples with different concentration levels were divided into inactivated group and non-inactivated group ，with 3 samples in each group. The inactivated plasma samples were heated at 56 ℃ for 30 min in metal bath with constant temperature. Non-inactivated group were not treated. After pretreating plasma sample of 2 groups，2-dimensional liquid chromatography was used to detect plasma concentration of the four drugs ；the difference of detection result between inactivated group and non-inactivated group were analyzed. RESULTS ：Plasma samples containing voriconazole，linezolid，vancomycin and teicoplanin were still stable after heating at 56 ℃ for 30 min in metal bath with constant temperature. Compared with non-inactivated group ，relative error of plasma concentration detection result of above 4 drugs were all lower than 15％ in low ，medium，high concentration mixed samples of inactivated group. CONCLUSIONS ：Plasma samples can be inactivated by heating at 56 ℃ for 30 min in metal bath with constant temperature ，when the plasma concentration of voriconazole，linezolid，vancomycin and teicoplanin are determined by 2-dimensional liquid chromatography.
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BACKGROUND: A proper virus inactivation procedure of medical bio-derived tissue repair material is very important to reduce the risk of virus infection and ensure the safety in the therapeutic process. OBJECTIVE: To elaborate different virus inactivation methods of allogeneic and xenogeneic tissue repair materials. METHODS: PubMed, Elsevier, CNKI, and WanFang databases were searched for relevant articles using the keywords of "allogeneic, xenogeneic, viral inactivation, disinfection, tissue repair biomaterial" in English and Chinese, respectively. RESULTS AND CONCLUSION: Virus inactivation methods can damage the performance of biological materials to different extents. For example, heat inactivation may produce permanent damage to the performance of heat-sensitive materials; γ-ray irradiation may result in the loss of mechanical properties and biologically active substances; acid/alkali method may also destroy the properties and structure of some materials intolerant to acid and alkali corrosion; and some reagent residues such as ethylene oxide, peracetic acid, and hydrogen peroxide may produce irritation to the body and even cause carcinogenic and teratogenic substances. Therefore, in enterprises and research institutions, the virus-killing effect and severity of damage to the material performance should be considered when the virus inactivation process is selected, and the use of existing production processes to verify the virus inactivation is recommended as much as possible.
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Objective To study the influence of residual methylene blue after plasma viral inactivation on the human immune cell function by using the peripheral blood mononuclear cell(PBMC) .Methods PBMC were isolated by adopting the Ficoll‐Hypaque density gradient centrifugation method and co‐cultured for 72 h in presence of specific T cell stimulating factors(Anti‐CD3/28 and Anti‐CD28) ,with or without different concentration of methylene blue .The culture supernatant was collected and detected the cyto‐kines secretion situation by ELISA .After 66 h culture ,CCK‐8 dye was added and continueously cultured for 4-6 h ,the prolifera‐tion was determined at A450 .Results The high‐concentration doses of methylene blue (1 .25 ,2 .5 ,5 μmol/L groups) had signifi‐cantly inhibiting effect on the proliferation of PBMC stimulated by Anti‐CD3/28(P< 0 .01) ,its OD value was decreased from 0 .897 ± 0 .385 to 0 .632 ± 0 .334 ,0 .524 ± 0 .254 and 0 .445 ± 0 .287 respectively ,showing certain dose dependent effect .The high concentrations of methylene blue (1 .25 ,2 .5 ,5 μmol/L groups) could down‐regulate interleukin(IL)‐17a ,IL‐10 and interferon (IFN)‐γ secreted by anti‐CD28 induced PBMC ,moreover showing a dose dependent effect .1 .25 ,2 .5 ,5 μmol/L methylene blue af‐fected the IL‐17a level secreted by PBMC from (406 ± 57)pg/mL descending to (276 ± 38) ,(192 ± 31) ,(134 ± 24)pg/mL respec‐tively ;affected PBMC to secrete IL‐10 ,its level was reduced from (184 ± 15) pg/mL to (132 ± 13) ,(110 ± 12) ,(42 ± 8)pg/mL ;af‐fected PBMC to secrete IFN‐γ,its level was deduced from (4 512 ± 187)pg/mL to (2 876 ± 143) ,(2 234 ± 153) ,(1 988 ± 112)pg/mL respectively .Conclusion High concentrations of methylene blue (≥1 .25 μmol/L ) has the significant inhibiting effect on the proliferation and cytokine secretion functions of PBMC .In other words ,the residual methylene blue concentration in viral inactiva‐tion plasma (≤0 .33 μmol/L) has no obvious effect on the immune function of PBMC ,but whether this concentration of methylene blue having the effect on human pure T cell immune function needs to be further evaluated and studied .
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Chemical disinfectants are generally used for virus inactivation and environment disinfection in biosafety laboratory, and the efficacy and evaluation of the disinfection are critical to ensure the laboratory biosafety.However, there is a current lack of applied standard to evaluate the virucidal efficacy of chemical disinfectants in our country.In this paper, a European Union standard“Method and Requirements of Virucidal Quantitative Suspension Test Method for Chemical Disinfectants Used in Human Medicine” was analyzed and a standard transformation scheme has been proposed.It is suggested that the model viruses should be increased from 3 to 6, including the surrogate viruses to substitute highly pathogenic viruses, and that the method to remove the residual chemical disinfectant and the calculation of 95%confidence interval should be incorporated into the standard.The suggestion will improve the scientific and operational standards related to disinfection and sterilization in biosafety laboratory in China.
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Plasma products are considered to be special medicinesderived from healthy human plasma .During 1980′s, events of transmission of human immunodeficiency virus through plasma products were frequently reported .Since then, ensuring the viral safety of plasma products has raised great concerns all over the world .So far, with decades of effort , most countries in the world have established rigorous systems with preventive measures to ensure the viral safety of plasma prod -ucts.These measures include control of source plasma , validated inactivation/removal of infectious agents , the adherence to current good manufacturing practices .Nevertheless , new infectious agents which may be threats to viral safety require continuous studies on appropriate countermeasures .
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Objective To study effect of virus inactivation/removal treated by solvent/detergent method and dry heating at 80℃, 72 h for inactivation in human coagulation factor Ⅷ.Methods Human coagulation factor Ⅷextracted from healthy human plas-ma were treated by solvent/detergent method and dry heating at 80℃, 72 h for inactivation .The virus inactivation effect was validated by adding the indicator virus ( PRV, Sindbis, HIV, EMCV, PPV).Results The methods could effectively inactivate lipid-enveloped and non lipid-enveloped viruses which could be used for virus inactivation /removal during human coagulation factor Ⅷexperiments , the residual amount of TNBP in production was less than one percent ten thousand (10 ppm), the residual Tween-80 concentration was less than one percent hundred thousand (100 ppm),which all met the safety standards .Conclusion and no significant change was ob-served in the activation and other indicators of human coagulation factor Ⅷ.
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BACKGROUND:Pasteurization is a perfect method for albumin virus inactivation, which may not be required for virus inactivation validation. However, there are no systematical reports concerning virus inactivation of hemoglobin blood substitutes. OBJECTIVE:To explore the effects of pasteurization on the physicochemical properties and biological function of hemoglobin blood substitutes. METHODS:Appropriate cord blood samples were taken fol owed by centrifugation, washing blood, rupture of membranes, stabilizer treatment. In the control group, the samples were placed in 55℃water bath, and when the temperature of hemoglobin solution reached (55±1)℃, a heat treatment began and lasted for 2 hours. In the pasteurization group, the samples were placed in 60℃water bath, and when the temperature of hemoglobin solution reached (60±1)℃, a heat treatment began and lasted for 10 hours. The heating process was under continues nitrogen protection. Then, the hemoglobin solution was placed in ice bath and cooled to below 4℃fol owed by low-speed centrifugation and filtration via microporous membrane, purification and viral inactivation thereby to obtain cord blood hemoglobin. RESULTS AND CONCLUSION:The products in the pasteurization group were al red clear liquid. There was no significant difference between the two groups in the yield, methemoglobin concentration, and oxygen-carrying capacity. The purification of the two groups was more than 98%. Two kinds of purification methods had no effects on the oxygen-carrying capacity of hemoglobin. Therefore, pasteurization method can replace thermosensitive purification method of 55℃, 2 hours. The pasteurization method wil not only ensure the physicochemical and biological properties of hemoglobin, but also achieve the purpose of virus inactivation.
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The aim of this study was to conduct a systematic review on the photoinactivators used in hemotherapy, with action on viral genomes. The SciELO, Science Direct, PubMed and Lilacs databases were searched for articles. The inclusion criterion was that these should be articles on inactivators with action on genetic material that had been published between 2000 and 2010. The key words used in identifying such articles were "hemovigilance", "viral inactivation", "photodynamics", "chemoprevention" and "transfusion safety". Twenty-four articles on viral photoinactivation were found with the main photoinactivators covered being: methylene blue, amotosalen HCl, S-303 frangible anchor linker effector (FRALE), riboflavin and inactin. The results showed that methylene blue has currently been studied least, because it diminishes coagulation factors and fibrinogen. Riboflavin has been studied most because it is a photoinactivator of endogenous origin and has few collateral effects. Amotosalen HCl is effective for platelets and is also used on plasma, but may cause changes both to plasma and to platelets, although these are not significant for hemostasis. S-303 FRALE may lead to neoantigens in erythrocytes and is less indicated for red-cell treatment; in such cases, PEN 110 is recommended. Thus, none of the methods for pathogen reduction is effective for all classes of agents and for all blood components, but despite the high cost, these photoinactivators may diminish the risk of blood-transmitted diseases.
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Nucleic Acids , Chemoprevention , Virus Inactivation , Blood SafetyABSTRACT
Purpose To test the virus inactivation effect of water bath method at 60 ℃ for 10 hours and alcohol treatment for 3 hours which was used in the production of urinary trypsin inhibitor(UTI).Methods Sindbis virus,Pseudorabies virus(PRV) and poliovirus1(PV1) were used as indicated viruses in this test.After being added separately into the UTI raw material in 10% proportion,the viruses were treated with water bath at 60 ℃ for 10 hours and alcohol for 3 hours and then the samples of UTI were taken to inoculate the cell line for assay of cytopathic effect.Results The water bath at 60 ℃ for 10 hours could inactive Sindbis,PRV and PV1 in more than(6.503±0.102)LgTCID_(50),(6.42±0.158) LgTCID_(50) and(6.587±0.061)LgTCID_(50) respectively,and alcohol treatment for 3 hours could inactive Sindbis,PRV and PV1 in more than(5.88±0.204)LgTCID_(50),(6.378±0.268)LgTCID_(50) and(5.963±0.118) LgTCID_(50) respectively.No cytopathic effect was found in the cell line which was inoculated with treated samples after blind passage for three generations.Conclusion The water bath method at 60 ℃ for 10 hours and alcohol treatment for 3 hours which were used in the production of UTI had good effects on virus inactivation and the inactivation efficiency on Sindbis,PRV and PV1 was more than 6 LgTCID_(50)/mL.
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The effective inactivation of microorganisms in drinking water by Ultraviolet (UV) irradiation is regarded as a new low-cost water treatment method shoeing high removal rate of relatively stable infectious virus particles including poliovirus. In the present study, we examined virus inactivation by UV in various water environments. Samples were collected from finished water and surface water, and tested for turbidity. UV dose of 18, 22, 30, 36 and 40 milli-Joule (mJ)/cm2 were used by combination of 2 mW/cm2 UV intensity and time of 9, 11, 15, 18 and 20 second. Depths of water were fixed at 0.37 cm and 8 cm, and virus titers were shown by plaque forming unit (PFU). Poliovirus was inactivated to 99.0% by 18 mJ/cm2 of UV dose in the condition of 0.08 Nephelometry Turbidity Unit (NTU) and 8 cm depth of water. Poliovirus at 30 mJ/cm2 of UV dose under the same condition was inactivated to 99.7%. Furthermore, Poliovirus at 56.60 NTU and 8 cm depth of water was inactivated to 92.0% and 98.5% by 18 mJ/cm2 and 30 mJ/cm2 of UV dose, respectively. The degrees of virus inactivation were dependent upon the UV dose, the turbidit, y and the depth of water. In conclusion, introduction of UV disinfections can be considered in drinking water purification systems in case reasonable engineering support is possible.
Subject(s)
Drinking Water , Nephelometry and Turbidimetry , Poliovirus , Shoes , Virion , Virus Inactivation , Water Purification , WaterABSTRACT
The effective inactivation of microorganisms in drinking water by Ultraviolet (UV) irradiation is regarded as a new low-cost water treatment method shoeing high removal rate of relatively stable infectious virus particles including poliovirus. In the present study, we examined virus inactivation by UV in various water environments. Samples were collected from finished water and surface water, and tested for turbidity. UV dose of 18, 22, 30, 36 and 40 milli-Joule (mJ)/cm2 were used by combination of 2 mW/cm2 UV intensity and time of 9, 11, 15, 18 and 20 second. Depths of water were fixed at 0.37 cm and 8 cm, and virus titers were shown by plaque forming unit (PFU). Poliovirus was inactivated to 99.0% by 18 mJ/cm2 of UV dose in the condition of 0.08 Nephelometry Turbidity Unit (NTU) and 8 cm depth of water. Poliovirus at 30 mJ/cm2 of UV dose under the same condition was inactivated to 99.7%. Furthermore, Poliovirus at 56.60 NTU and 8 cm depth of water was inactivated to 92.0% and 98.5% by 18 mJ/cm2 and 30 mJ/cm2 of UV dose, respectively. The degrees of virus inactivation were dependent upon the UV dose, the turbidit, y and the depth of water. In conclusion, introduction of UV disinfections can be considered in drinking water purification systems in case reasonable engineering support is possible.
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Drinking Water , Nephelometry and Turbidimetry , Poliovirus , Shoes , Virion , Virus Inactivation , Water Purification , WaterABSTRACT
9.9Log10 bacteriophage R 17 Conclusion Alkylation of the phenothiazine ring increased the potential of inactivating virus and decreased the damage to red blood cells? M007 might be an alternative phenothiazine dye that can be used to inactivate viruses in red blood cells?
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Objective To study the degradation of hepatitis C virus(HCV) genome before and after methylene blue photochemistry(MB-P) treatment of the plasma.Methods MB was added to HCV positive plasma to a final concentration of 1.0?mol/L.The plasma was then exposed to 30000 Lux fluorescence.Plasma samples were then collected at different times of exposure.Real-time PCR was used to quantitatively study the course of the HCV-RNA degradation.The whole genome was screened for integrity by RT-PCR with 8 pairs of specific primers which targeted sequential overlapping sections of HCV genome.Results Results of real-time PCR showed that the copy number of HCV-RNA was continuously decreasing during MB-P treatment.RT-PCR results showed that the reactivity of different sections of the HCV genome to MB-P was significantly different and indicated that the 5' end and 3' end were more stable. Conclusion MB-P treatment could degrade HCV RNA with various sensitivities to different sections of the genome.RNA degradation may play an important role in plasma virus inactivation.Detection of HCV genomic RNA might be clinically useful to monitor the process and efficiency of virus inactivation.
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Objective To observe the effects of methylene blue photochemical(MB-P) inactivation of human cytomegalovirus in blood products.Methods Plasma and red blood cell suspensions containing 10% HCMV and 5?mol/L MB were illuminated by fluorescent light of 38000 Lux for 1 hour, HCMV was used as model viruses for validation of virus inactivation.Results The 50% tissue culture infective dose (TCID_ 50 ) contained in plasma and red blood cell suspensions decreased by 4~6 times.Conclusion MB-P treatment is effective in inactivating the infectivity of HCMV in plasma and red blood cell suspension.
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The preliminary results of inactivation of model parvovirus M13Mp 18 in plasma by long-waveUVA irradiation combined with psoralen derivatives were described.At 8-MOP concetration of300?g/ml plasma and UVA intensity of 11.5mW/cm~2,the UVA irradiation for 30～120 min couldresult in virus inactivation of 10~(5～9) infectious dose/ml.Quenchers were used to reduce the damage ofUVA to proteins in order to improve the clotting factor recovery after irradiation.2mmol/Lglutathione,or 2mmol/L glutathione with 2 mmol/L mannitol could significantly improve the Frecovery after irradiation.
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Objective To research the effect of Hydroxyl radical(type Ⅰ mechanism)and singlet oxygen(type Ⅱ mechanism)on virus inactivation and red cell cytotoxicity induced by DMMB and M007 phototreatment.Methods Hydroxyl radical quenchers mannitol and glycerol,singlet oxygen quenchers sodium azide and tryptophan were employed to investigated their ability to inhibit the inactivation of bacteriophage Phi6 and red cell cytotoxicity by induced DMMB and M007 phototreatment.Results Singlet oxygen quenchers could inhibit the inactivation of bacteriophage Phi6 obviously, whereas hydroxyl quenchers hardly affect the inactivation of bacteriophage Phi6.Both type of quenchers could reduce the hemolysis caused by CMMB or M007 phototreatment.Conclusion Viral inactivation of DMMB and M007 phototreatment is mainly through type Ⅱ mechanism, both type I and type Ⅱ mechanisms contribute to red cell cytotoxicity.